LYSYL-TRNA SYTHETASE AND APXN SYNTHESIS IN E COLI

大肠杆菌的赖氨酰-tRNA合成酶（LysRS）系统由以下组成： 两个差异调节基因一个基因lysS是组成型的， 第二个是lysU，是可诱导的。后者是已知的细胞应激基因， 并且其表达在热休克时被诱导。的大肠大肠杆菌氨酰- tRNA合成酶，LysRS后接苯丙氨酰-tRNA合成酶（PheRS） 已经在体外被证明是最有效的合成 腺苷酰化二核苷寡磷酸（ADO），核苷酸家族 其原型是AP 4A。ADO的细胞内浓度 当E.大肠杆菌、其他细菌或真核细胞， 经受诸如热冲击、氧化剂或重金属的应力 离子。重要的是确定是否在E.大肠杆菌中，每个LysRS的产物 基因和/或PheRS是体内合成的主要催化剂 的ADO。E. lysS基因、lysU基因缺陷的大肠杆菌突变体，或 pheS基因（pheRS）以及双LysS LysU突变体和三重LysS LysU突变体。 LysS LysU PheS三重突变体现在可在本实验室获得。这使得 可以确定在细胞应激时，单个突变细胞，或 具有双重或三重突变的菌株将具有 ADO，或者如果每个LysRS基因产物和PheRS可能优先 合成ADO.这一发现表明，细胞活力的丧失， 压力与缺乏任何ADO的合成相关， 为研究这些化合物的细胞作用指明了方向。这 由于ADO是在所有细胞中制备的， 被怀疑在DNA合成中发挥作用。此外，这项工作 有可能建立一个基本的体内作用， 非氨酰化tRNA合成酶 存活率将通过测定之前和之后的菌落形成单位来评估， 在施加压力之后。 核苷酸将用32 P标记， 在应力之前，将在适当的时间取样， 施加应力（热、氧化或重金属）。的 核苷酸将通过二维薄层色谱分离 (TLC)使用适当的标准品，放射自显影，切下平板， 通过闪烁计数定量。

The lysyl-tRNA synthetase (LysRS) system of Escherichia coli consists of two differentially regulated genes. One gene, lysS, is constitutive, and the second, lysU, is inducible. The latter is a known cell stress gene, and its expression is induced upon heat shock. Of the E. coli aminoacyl- tRNA synthetases, LysRS followed by phenylalanyl-tRNA synthetase (PheRS) have been shown in vitro to be the most efficient in synthesizing adenylylated dinucleoside oligophosphates (ADO), a family of nucleotides of which the prototype is AP4A. The intracellular concentration of the ADO rises markedly when E. coli, other bacteria, or eukaryotic cells are subjected to stresses such as heat shock, oxidizing agents, or heavy metal ions. It is important to determine if in E. coli the product of each LysRS gene, and/or PheRS are the predominant catalysts for the in vivo synthesis of the ADO. E. coli mutants defective in the lysS gene, the lysU gene, or the pheS gene (pheRS) as well a double LysS LysU mutants, and a triple LysS LysU PheS triple mutant are now available in this lab. This makes it feasible to determine whether upon cell stress, a singly mutant cell, or a strain with a double or triple mutation will have a normal output of ADO, or if each LysRS gene product and PheRS might preferentially synthesize a subset of ADO. The finding that a loss of cell viability upon stress correlates with a lack of synthesis of any ADO will potentially point the way, to studies on the cellular role(s) of these compounds. This would be of fundamental importance since ADO are made in all cells, and are suspected of having a role in DNA synthesis. Additionally this work has the potential to establish a fundamental in vivo role for some aminoacyl-tRNA synthetases other than for aminoacylation of tRNA cell survival will be assessed by determining colony forming units before and after application of a stress. The nucleotides will be labeled with 32P, prior to a stress, and samples will be taken at appropriate times upon application of the stress (heat, oxidation, or heavy metal). The nucleotides will be resolved by two-dimensional thin layer chromatography (TLC) using appropriate standards, autoradiographed, cut off the plate and quantitated by scintillation counting.